We have studied myosin phosphorylation during contraction of tracheal smooth muscle (TSM). We have found that myosin is phosphorylated when these muscles are contracted with methacholine and that the exten of phosphorylation and the tension generated are dependent on the concentration of methocholine used to contract TSM. We have also studied myosin dephosphorylation and cAMP formation during relaxation of TSM. The data indicate that myosin dephosphorylation, but not cAMP formation, is essential for relaxation of TSM.